We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on lipid and retinoid metabolism pathways, differentiation/dedifferentiation pathways, and protection against oxidative stress. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration (AMD) and may be regulated by retinoic acid (RA). RA affects many cellular functions including cell growth, differentiation, and apoptosis. Synthetic analogs of retinoic acid also have significant effects on cellular function and are being studied. Also, the role of signaling pathways in RPE differentiation and de-differentiation is an important focus of our research. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In addition, given the likely importance of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in the response of RPE cells to various signals, we are interested in determining changes in miRNA expression in RPE cells due to agents with which they are treated in our experiments. In the past year we have made progress in the following areas: 1) Stearoyl-CoA desaturase (SCD) regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in ARPE-19 cells. Increased expression of BiP/GRP78, ATF4 and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR-41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases such as AMD. A manuscript describing this work was published in this reporting period. 2) We continued our work on the role of miRNAs in regulating the inflammatory response of the retinal pigment epithelium (RPE), implicated in the pathogenesis of age-related macular degeneration. The role of microRNAs miR-146a and miR-146b-5p in regulation of the inflammatory process induced by the proinflammatory cytokines interferon (IFN)-&#947;, tumor necrosis factor (TNF)-&#945;, and interleukin (IL)-1&#946; in human RPE cells has been studied. We are also conducting experiments to modulate the effect of proinflammatory cytokines on the inflammatory response using anti-inflammatory agents such as resveratrol. These experiments have been extended to long-term mature confluent ARPE-19 monolayers to evaluate the effect of proinflammatory cytokines on miRNA expression in differentiated RPE cells. 3) We have expanded a study to understand the mechanisms underlying dedifferentiation of RPE cells in primary culture and redifferentiation in the ARPE-19 RPE cell line. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research. On the one hand, using a cocktail of factors induced pluripotent stem (iPS) cells can be differentiated into cells sharing many aspects of RPE phenotype, and by rigorous culture methods, fetal RPE cells can be differentiated to retain or acquire aspects of native phenotype. On the other hand, explanted native RPE cells will lose important aspects of their RPE phenotype after a short time in culture. The various cell lines, such as the commonly used ARPE-19, do not have most native phenotypic features under common culture methods. What are the mechanisms regulating such gain or loss? Do mechanisms like epithelial-mesenchyme transition play a role in this process? We are particularly interested in the long-known but poorly understood loss by immortalized and primary RPE cells of expression of visual cycle enzymes. Understanding the mechanism underlying this down-regulation could be useful in ensuring that iPS-derived cells used for human transplant are fully competent to fulfill their intended role in restoring RPE function in treated eyes. Our experimental paradigm focuses i) on the loss of visual cycle phenotypic competence by adult bovine RPE cells explanted into primary culture, and ii) the regain of phenotypic competence by careful culture of relatively early passage ARPE-19 cells. Using these models we are analyzing expression of visual cycle and genes of other pathways and will correlate these to changes in gene regulation, RNA transcript expression and microRNA expression patterns. Striking changes in phenotype (cell morphology and melanization), gene expression, and biochemistry have been observed in ARPE-19 cells grown for 4 months or more, and indicate that ARPE-19 retains the plasticity to return to native-like RPE phenotype. The study is still ongoing. 4) We continued analysis of post-transcriptional modifications of IRBP. In this reporting period we have continued to study whether phosphorylation of IRBP occurs in vivo. An HPLC method to purify phosphopeptides has been established and these are being analyzed by mass spectrometry. We continue to collaborate within the LRCMB and with other laboratories and sections (LI, Molecular Structure and Functional Genomics), as well as with extramural labs in the analysis of retinoid and other compounds. A manuscript on the latter was published during this reporting period.